The central goal of this project is to comprehensively identify post-transcriptional regulators of gld-1, which encodes a conserved RNA-binding protein that acts to promote the decision for germline stem cells to differentiate and enter meiosis. Post-transcriptional regulation of gene expression contributes to the decision of a stem cell to either maintain the stem cell fate or to differentiate. In the germline stem cells of C. elegans, posttranscriptional repression of gld-1 contributes to maintenance of the stem cell fate whereas translational activation of gld-1 promotes meiotic entry. Multiple post-transcriptional regulators of gld-1 are known: FBF-1 and FBF-2 are RNA-binding proteins that directly repress gld-1, whereas the RNA binding protein GLD-3, the poly-A polymerases GLD-2 and GLD-4, and NOS-3 activate gld-1. However, genetic analysis of these genes indicates other regulators must also contribute to gld-1 repression and activation in the distal germline. We propose to use RNA-based affinity purification of proteins that bind to the gld-1 mRNA in vitro and in vivo, followed y mass spectrometry to identify these bound proteins. This unbiased approach will generate a comprehensive catalogue of proteins that bind to the gld-1 mRNA during translational repression and during translational activation. Identified proteins will be validated for a role in gld-1 repression or activation genetically. A genetics approach will also be used to determine if the microRNA pathway represses gld-1 in germline stem cells, which was hypothesized based on recent computational, genetic, and functional data. Identification and validation of new gld-1 regulators is necessary to generate a molecular model for how gld-1 switches from being repressed to being activated as part of the decision of germline stem cells to enter meiosis. Understanding this switch will provide important insight into how stem cells initiate intracellular events to execute the decision to differentiate.